The direct Coombs test (also known as the direct antiglobulin test or DAT or DAGT) is used to detect if antibodies or complement system factors have been bound to erythrocyte or red blood cells (RBC) surface antigens in vivo. Such bound antibodies are associated with various diseases in which an immune mechanism is attacking the patient's own RBCs. This mechanism could be autoimmunity alloimmunity or a drug-induced immune-mediated mechanism. In more detail such diseases include:
Examples of Alloimmune Haemolysis
Hemolytic disease of the newborn (also known as HDN or erythroblastosis fetalis)
Rhesus D hemolytic disease of the newborn (also known as Rh disease)
ABO hemolytic disease of the newborn (the indirect Coombs test may only be weakly positive)
Anti-Kell hemolytic disease of the newborn
Rhesus c hemolytic disease of the newborn
Other blood group incompatibility (RhC, Rhe, RhE, Kid, Duffy, MN, P and others)
Alloimmune haemolytic transfusion reactions
Examples of Autoimmune Haemolysis
Warm antibody autoimmune hemolytic anemia
Idiopathic
Systemic lupus erythematosus
Evans' syndrome (antiplatelet antibodies and haemolytic antibodies)
Cold antibody autoimmune hemolytic anemia
Idiopathic cold hemagglutinin syndrome
Infectious mononucleosis
Paroxysmal cold hemoglobinuria (rare)
Drug-induced immune-mediated haemolysis
Methyldopa
Penicillin (high dose)
The complement system is composed of a number of small proteins found in the blood, which co-operate with the antigen-antibody interaction to kill target cells. Over 20 proteins and protein fragments make up the complement system.
Conventionally the DAT test has been carried out as an agglutination test in a test tube. More recently this test has also been carried out using agglutination microplate and gel technology. The test however, is still somewhat cumbersome and automated read-out of the results can be problematic.
More recently it has been found that ABO blood typing can be successfully carried out using non-agglutination protein microarrays, in which an immobilized antibody binds to an antigen on the surface of the RBC, and the presence of RBCs so immobilized is detected (J S Robb et al 2006). It has further been found that antibody microarray technology can be used to phenotype erythrocytes by detecting complex mixtures of antigens on cell surfaces (C J Campbell et al 2006). The antigens are both sugar antigens, which tend to be well presented and easily accessible, and peptide antigens, which are epitopes of transmembrane proteins and therefore buried and held more closely to the cell surface, and these were successfully differentiated using the correct choice of antibodies.
We have now surprisingly found that RBCs coated with antibody and/or complement (protein) can withstand the required processing and remain ‘sensitised’ (coated) with said antibody or complement bound to said RBCs, and that microarray technology can be used to detect antibodies and/or complement present on the surface of RBCs, thereby providing a test which is a much more efficient and an effective alternative to conventional DAT testing, and which can, moreover, be readily integrated into a single microarray with other tests important in blood processing—including blood grouping phenotyping for multiple antigens on the surface of the RBC.